Recombinant proteins having haemostatic activity and capable of inducing platelet aggregation

ABSTRACT

Disclosed herein are recombinant proteins capable of inducing platelet aggregation and uses thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a §371 National Stage Application ofPCT/EP2009/062271 filed Sep. 22, 2009, which claims priority to FrenchApplication No. 085 64 23 filed Sep. 24, 2008.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to recombinant proteins composed of theassembly of the peptide sequences described for interacting withplatelet collagen receptors. These proteins have a pro-aggregatingactivity, independent of the formation of a triple helix. They can beproduced in bacteria and in mammalian cells.

2. Description of Related Art

Collagens are the principal structural components of the extracellularmatrix of all multicellular organisms. They are a family of proteinscomposed of 28 different types that play a role during development andin tissue homeostasis. They are capable of being assembled into varioussupramolecular structures in the form of fibrils, microfibrils ornetworks.

Collagens have the common characteristic of containing one or moredomains that have a triple helix structure formed of three polypeptidechains, or α chains, coiled together. As all three are amino acids, thischaracteristic is enabled by the presence of a glycine in the helicoidalmotifs which consist of repeated G-X-Y sequences where X is often aproline and Y a hydroxyproline. This hydroxyproline is essential to thestabilisation of the triple helix and is characteristic of collagens.The proline residues are hydroxylated primarily by prolyl-4-hydroxylase(P-4-H) into 4-hydroxyproline. There is a second hydroxylase,prolyl-3-hydroxylase, which enables the hydroxylation of proline when itis in position X, whereas in position Y a proline hydroxylated by P-4-His already found. In a collagen molecule, the alpha chains can beidentical or different.

Collagen molecules are formed of helicoidal domains (or collagenousdomains) flanked by non-helicoidal domains, called N- and C-propeptides.Recognition of the three α chains that form a molecule and the start oftheir assembly are under the control of the C-terminal end(C-propeptide). This process is carried out at the endoplasmicreticulum. Subsequently, the N- and C-propeptides are excised duringcollagen maturation, leaving short non-helicoidal sequences,telopeptides.

Collagens play an important role at the vessel walls by maintainingtheir integrity and their elasticity. This wall is formed of type-Icollagen and type-III collagen, which are fibrillar, and of type-IVcollagen, in network form. It should be noted that type-III collagen isalso strongly expressed at atheromatous plaques. Furthermore, collagensare capable of modulating the functions of certain cells by directinteraction with specific cellular receptors. Thus, collagens, mainlytypes I and III, are powerful activators of platelet function.

Type-III collagen is a homotrimer composed of three α1 chains arrangedin a triple helix. Two G-P-P triplets (potentially hydroxylated),present at the C-terminal end of the α1 chains, are then sufficient andnecessary to the nucleation and the folding of the triple helix (Bulleidet al., EMBO J. 1997 Nov. 17; 16(22): 6694-701). On the other hand,these two triplets are not sufficient to enable the initial combining ofthe three chains. It is interesting to note that Bulleid et al. (1997)observed greater efficiency of triple helix formation when threetriplets are maintained, and this efficiency is even greater than forthe wild molecule. Several proteins are involved in the assembly processof monomeric chains: HSP47 (heat shock protein) and PDI (proteindisulfide isomerase). C-propeptide, located at the C-terminal end ofmonomeric chains, appears involved in the alignment of α chains and theformation of the disulfide bridges necessary to the stabilisation of theprotein (Bulleid et al., EMBO J. 1997 Nov. 17; 16(22): 6694-701). ThisC-propeptide is thus necessary only to ensure the combining of themonomeric chains. Various points should be remembered in relation to it:

-   -   It consists of a discontinuous sequence of 15 amino acids which        determines the specific standard assembly of α1 chains (Hulmes D        J, J Struct Biol. 2002 January-February; 137(1-2): 2-10).    -   A coiled-coil domain located at the beginning of the        C-propeptide is involved in the trimerisation of collagen III        (Bulleid et al., EMBO J. 1997 Nov. 17; 16(22): 6694-701). This        domain consists of four heptapeptides (McAlinden et al., J Biol        Chem. 2003 Oct. 24; 278 (43): 42200-7).    -   It contains eight cysteine residues which enable the formation        of intra- and intercatenary disulfide bridges.

The presence of disulfide bridges between chains at the C-telopeptide orC-propeptide is not necessary for the combining of the chains and theformation of the triple helix (Bulleid et al., Biochem J. 1996 Jul. 1;317(Pt 1): 195-202). It should be noted that the experiments areundertaken with the N-propeptide. The notions of trimer and of alphatriple helix seem paradoxically independent. In the absence ofN-propeptide, it seems judicious to leave either cysteine 2 of theC-propeptide, or the two cysteines of the C-telopeptide. This lattermotif, called the knot sequence (GPCCG), enables the formation ofdisulfide bridges only by virtue of a preliminary phenomenon of assemblyand folding of the α1 chains (Boudko and Engel, J Mol Biol. 2004 Jan.30; 335(5): 1289-97).

Whether of traumatic origin or the consequence of atherosclerosis,damage to the arterial wall is accompanied by the destruction of thevascular endothelium and the exposure of thrombogenic components such ascollagen. This is followed by adhesion of platelets at the damaged site,in contact with surfaces rich in collagen or collagen fragments, theiractivation and the formation of a thrombus. The adhesion andstabilisation of the platelets in contact with this collagen are enabledby multiple interactions, of high affinities, between receptors presentat the surface of the platelets and the collagen. This adhesion canoccur indirectly following the binding of the A1 domain of the vonWillebrand factor (vWF) with the platelet complex formed of theglycoproteins (Gp) GpIb-V-IX, itself bound to collagen by its A3 domain,or by direct interaction between a platelet receptor and collagen.Several receptors can bind collagen molecules at highly specific peptidesequences, namely integrin α2β1, which plays an important role in thestabilisation of the platelet in contact with collagen, and GpVI,considered as the most important receptor for platelet activation.Another receptor, TIIICBP (type-III collagen-binding protein), has beendescribed as capable of binding collagen directly.

Blood vessel flow rate is a major determinant that defines the type ofplatelet collagen receptors recruited. At an elevated flow rate, theplatelets interact with collagen via vWF via the GpIb receptor and thenthey are activated by binding via GpVI; integrin α2β1 only intervenes asa stabiliser of the platelet-collagen bond. At a low flow rate, theplatelets bind to collagen via integrin α2β1, followed by binding toGpVI, thus leading to their activation, which is the step that preparesplatelet aggregation. Similarly, other platelet collagen receptors areinvolved such as TIIICBP. In all cases, GpVI plays a major role inplatelet activation.

Platelet adhesion is a process that is now dissociated from plateletactivation. Indeed, many peptides, corresponding to short peptide motifsof collagen, are capable of inducing the adhesion of platelets withoutleading to their activation. Nevertheless, some of these peptides havethe capacity to induce platelet activation and exhibit a so-calledpro-aggregating activity. Their triple helix structure seems to be anessential prerequisite for this activity. It should be noted that whensome of these peptides remain in monomeric form, they exhibit ananti-aggregating activity by a mechanism which remains to be identifiedbut which could be the occupation of sites that become unavailable fornative collagen.

Most of the work aimed at identifying the peptide sequences involved inthe adhesion of platelets with type-III collagen use the fragmentα1(III) CB4 corresponding to the digestion fragment by CNBr exhibitingthe highest aggregating activity.

Various peptide motifs have been described in the past few years asbeing capable of binding and activating platelets. They have been testedin the form of peptides of two possible natures:

-   -   similar to collagen, formed by the repetition of conserved GPO        motifs repeated n times, not present in the native sequence of        type-III collagen;    -   or corresponding to peptides formed of peptide motifs present in        the α1(III) CB4 sequence, principally obtained by chemical        synthesis (Farndale et al., Biochem Soc Trans. 2008 April; 36(Pt        2): 241-50).

Integrin α2β1 is a receptor for collagens, laminin and other ligands invarious cell types including endothelial cells and platelets. α2β1 bindscollagen via its domain I at the peptide motifs present in the sequenceof fibrillar collagens. Various motifs have been described with thehighest affinity for GFOGER present at the α1 chain of type-I collagen.For type-III collagen, and contrary to type-I collagen, it seems thatseveral GXYGER motifs are necessary for optimal binding, with GLOGER,GMOGER, GROGER and GAOGER as principal motifs (Kim et al., J Biol Chem.2005 Sep. 16; 280(37): 32512-20). Other motifs such as GLOGEN and GLKGENhave been described but exhibit low affinity for α2β1 (Raynal et al., JBiol Chem. 2006 Feb. 17; 281(7): 3821-31). It should be noted that allof the peptides synthesised from these motifs exhibit an adhesionactivity when they are organised in the form of a triple helix.

Glycoprotein VI (GPVI) is a 60-65 kDa type-I transmembrane glycoproteinbelonging to the immunoglobulin (Ig) superfamily. It is constitutivelyexpressed on the surface of platelets in the form of a non-covalentcomplex with the γ chain common to Ig receptors (FcRγ). The peptidesdescribed as interacting with this receptor are peptides similar tocollagen formed from the repetition of 4 to 10 GPO triplets (Morton etal., Biochem J. 1995 Mar. 1; 306(Pt 2): 337-44 and Smethurst et al., JBiol Chem. 2007 Jan. 12; 282(2): 1296-304). Although comprising 10% GPOmotifs, the maximum number of repetitions of this triplet does notexceed three in the native sequence of type-III collagen. It should befurther noted here that the formation of the triple helix or of apolymeric form, obtained chemically following a modification of thecysteine or lysine residues, is essential to confer on these peptides apro-aggregating activity. Moreover, the presence of a hydroxyproline inposition 3 is essential to this activity. Conversely, when they are inmonomeric form, these peptides exhibit an anti-aggregating activity(Asselin et al., Biochem J. 1999 Apr. 15; 339 (Pt 2): 413-8). Recently,Jarvis et al. identified the peptide motif in the type-III collagensequence that exhibits the highest adhesion activity. It is composed ofresidues GAOGLRGGAGPOGPEGGKGAAGPOGPO located at amino acids 523 to 549of α1(III) CB4 (Jarvis et al., Blood. 2008 May 15; 111(10): 4986-96).This peptide motif is composed of three GPO, which are not consecutiveand which seems to be the minimal number necessary for properinteraction with GpVI.

Indirect interaction between the platelet receptor GpIb and collagen isdependant on vWF, which is a plasma multimeric protein secreted byendothelial cells and platelets in response to vascular damage orfollowing an increase in parietal pressure. It plays a major role in therecruitment of platelets at damaged sites of vascular territoriessubjected to elevated flow rates. This factor is composed of threedomains named A1, A2 and A3. It binds collagen via its A3 domain andbinds the GpIb receptor by its A1 domain. Type-III collagen seems tohave a single site, of high affinity for the A3 domain of vWF, which isalso present in type-II collagen. This peptide motif is located betweenamino acids 403 and 413 and is composed of GPRGQOVMGFO with certainamino acids critical for binding vWF (Lisman T et al., Blood. 2006 Dec.1; 108(12): 3753-6). Verkleij et al. identified as a potential bindingsite amino acids 541 to 558 composed of GAAGPOGPOGSAGTOGLQ (Verkleij etal., Blood. 1998 May 15; 91(10): 3808-16). This peptide motif is locatedbetween the vWF motif described by Lisman et al. and the GMOGER integrinα1β2 binding motif.

The Fauvel-Lafève team described the existence of an octapeptide,KOGEOGPK, located between amino acids 655 and 662 of the fragmentα1(III) CB4. The platelet receptor recognised by this octapeptide hasbeen identified and named TIIICBP (type-III collagen binding protein)(Monnet E et al., J Biol Chem. 2000 Apr. 14; 275(15): 10912-7). Thisoctapeptide is capable of inhibiting the interaction of platelets withtype-III collagen, but not with type-I collagen, under both static andflow conditions. More recently, Pires et al. showed that thisoctapeptide has an inhibiting activity on aggregation only when it is inhomotrimer form. On the contrary, its structure in triple helix form byadding at these ends cysteines and GPP gives it a pro-aggregatingactivity (Pires et al., Eur J Med Chem. 2007 May; 42(5): 694-701).

The biological and ultrastructural properties of collagens, and notablytheir capacity of binding to membrane receptors, open a broad field ofapplications because of their multiple roles in tissues. However, theseapplications only have meaning if it is possible to have homogeneouspreparations of these collagens in large quantities and in areproducible way.

Two methods of production were used to this end. The work anddevelopments conducted in these two areas are generally highly targetedfor a given application. Thus, chemical synthesis can produce shortpeptides that represent only a tiny part of the protein, in generalpeptide motifs of interest. Its principal application relates tohaemostasis and more precisely to modulation of the bond betweencollagen and platelets.

However, these peptides have in general only one of the activitiessought and this can depend on the three-dimensional conformation of thepeptide and notably of the formation of a triple helix. Thus, there is asignificant need for the development of functionalised recombinantcollagen proteins that can be produced by biological systems offeringhigh productivity. A major obstacle is the difficulties of productionand purification of these collagen-derived proteins because of theirtendency to aggregate and to adhere.

To date, short peptide sequences (fewer than 50 amino acids) have allbeen produced by chemical synthesis and not via cellular productionsystems (Farndale et al., Biochem Soc Trans. 2008 April; 36(Pt 2):241-50). Moreover, these motifs have been synthesised in an isolatedway. Conversely, the synthesis of recombinant type-III collagen,exhibiting platelet binding activity, has been carried out via cellularproduction systems and it is the totality of the sequence, for exampleproα1(III), which has been used. The objective sought is the synthesisof a whole procollagen capable of being organised into a triple helix(WO 9 307 889). These recombinant collagens have in principle multipleapplications, namely those of the complete collagen sequence.

It is thus a question of synthesising smaller proteins of simplerstructure that are thus less demanding in terms of production but thatpreserve the biological activities of interest of the native protein.

SUMMARY OF THE INVENTION

The invention thus relates to recombinant proteins possessing the motifsnecessary to obtain a pro-aggregating activity, synthesised in the formof a monomer incapable of being structured into the form of a triplehelix. The impossibility of forming this triple helix is the consequenceof the absence of: I) α chain recognition motifs between them, II)non-helicoidal collagen N- and C-terminal domains as well as III) twoGPO triplets necessary to the priming of the triple helix. Surprisingly,pro-aggregating activity is obtained in the absence of the formation ofa triple helix.

The Applicant has shown in a surprising way that it is possible to bringtogether three types of peptide sequences within a single monomericprotein having pro-aggregating activity.

The recombinant proteins of the present invention thus bring together:

-   -   peptide motifs with at least one repetition of four GPO        triplets,    -   peptide sequences described as having binding activity to the        various platelet receptors present in the native collagen        sequence,    -   binding sequences between these motifs formed of the repetition        of GXY triplets.

The production of these proteins by bacteria and mammalian cells makesit possible to have in large quantities these new-generation recombinantcollagens, fruits of the combination of peptide sequences similar tocollagen and sequences present in the native form of collagen. Thesetypes of proteins do not require favouring cells that do not producecollagens constitutively as host cells for this production. Indeed,their sequence does not allow recognition of the alpha chains of variousnative collagens. On the contrary, it is necessary to favourcollagen-producing cells that express key enzymes of collagen maturationsuch as prolyl-4-hydroxylase (P4H) and HSP47. Current recombinantcollagen production systems to the contrary use cells that do notnaturally produce collagen (Olsen et al., Adv Drug Deliv Rev. 2003 Nov.28; 55(12): 1547-67 and Ruggiero and Koch, Methods. 2008 May; 45(1):75-85). This involves cotransfecting the gene coding for enzymes such asP4H.

The recombinant proteins according to the invention have manyadvantages, including:

-   -   bringing together motifs of interest within small proteins that        are easier to produce using cellular production systems,    -   pro-aggregating activity in the absence of a triple helix        structure,    -   the absence in their sequence of collagenase recognition sites        but the presence of HSP47 recognition sites, enabling their        production by collagen-producing cells,    -   their biological activity shown by reference tests (platelet        aggregation tests) which are used for monitoring current        antiplatelet treatments and for exploring platelet functions.

The biological properties of the recombinant proteins according to theinvention make it possible to envisage many applications.

The first application relates to the use of these proteins as adiagnostic tool in the exploration of thrombotic disease: plateletaggregation tests, in vitro thrombosis models and evaluation of theeffectiveness of current antiplatelet treatments (aspirin, clopidogrel)and future treatments. The proteins produced are derived from humanproteins and have in their sequence binding sites for certain plateletreceptors described for their role in platelet adhesion and activation.Current tests use collagens of animal origin whose sequences do not haveperfect homology with those of humans. Furthermore, variation of thenumber and location of platelet receptor interaction sites between thevarious proteins makes it possible to propose more relevant targetvalidation tests. There are peptide fragments that make it possible towork more specifically on each platelet receptor but their method ofproduction is different from that of the invention, with chemicalsynthesis versus natural production by cells (importance ofpost-translational modifications).

The present invention can play a part in the composition of haemostaticbandages (thus strengthening on-site recruitment of platelets in thecase of vascular lesion), or of haemostatic adhesives to prevent therisk of bleeding (surgical context). Products are currently on themarket, but with a collagen of animal origin.

Another application is the use of these proteins to activatecicatrisation. Cicatrisation of certain wounds requires the on-sitemigration, adhesion and differentiation of certain cell populations,including platelets. These processes are dependant on the surfaceintegrins of these cells. Certain peptide motifs present in thestructure of the inventive proteins are thus capable of interacting withthese integrins by promoting cicatrisation.

In a very close field, these recombinant proteins could be proposed inthe composition of certain dermatological creams.

Another application of proteins that inhibit platelet function is thetreatment of atherothrombosis. Platelet receptors are therapeutictargets with a future for fighting complications of atherothromboticdisease. To date, there is no molecule capable of blocking the initialphase of platelet adhesion in the subendothelium. The various proteinstructures of the inventive proteins enable their interaction with thesereceptors leading to platelet anti-adhesion effects (site masking)and/or platelet pro-adhesion effects. The various effects can beobtained with the same protein by modulating its physicochemicalproperties.

This invention thus provides a biological reagent with numerousapplications that can play a part in the composition of kits: toevaluate platelet functions, to activate cell differentiation for thediagnosis of haematological dysfunctions, to detect by imaging areas offibrosis. This invention can also enter in the composition ofhaemostatic bandages for the on-site recruitment of platelets (in thecase of vascular lesion), haemostatic adhesives to prevent the risk ofbleeding, or dermatological creams.

DESCRIPTION OF SEQUENCES

SEQ ID No. 1: Coll-III-like recombinant protein

SEQ ID No. 2: Polynucleotide coding for Coll-III-like recombinantprotein

SEQ ID Nos. 3-6: Primers

DESCRIPTION OF THE INVENTION

The invention relates to isolated polypeptides that have the sequence ofthe polypeptide of SEQ ID No. 1 or the polypeptide of position 25 toposition 152 of SEQ ID No. 1.

The invention also relates to isolated polypeptides with at least 70%identity over their entire length with the polypeptide of SEQ ID No. 1or with the polypeptide of position 25 to position 152 of SEQ ID No. 1,and capable of inducing an aggregation of human blood platelets greaterthan 30% in a platelet aggregometer at 37° C. with stirring of 1000 rpm.

In a preferred embodiment, these polypeptides comprise the followingpeptide motifs:

-   -   GX₁X₂GER wherein X₁ and X₂ represent independently an amino acid        selected from A, R, N, D, Q, E, G, H, I, K, M, F, P, S, T, W, Y,        V and O;    -   (GPX₃)n with n between 4 and 10 and X₃ representing P or O;    -   GPRGQX₄GVMGFX₅ where X₄ and X₅ represent independently P or O.        P is proline and O is hydroxyproline.

In another preferred embodiment, these polypeptides comprise thefollowing peptide motifs:

-   -   GAPGER,    -   KPGEPGPK,    -   (GPP)n with n between 4 and 10,    -   RGD.

The invention also relates to an isolated polynucleotide characterisedin that it codes for a polypeptide according to the invention.

In a preferred embodiment, the polynucleotide has the sequence of thepolynucleotide of SEQ ID No. 2.

The invention also relates to an antibody binding specifically to thepolypeptide of SEQ ID No. 1.

The invention also relates to expression cassettes comprising in thedirection of transcription:

-   -   a functional promoter in a host organism,    -   a polynucleotide according to the invention,    -   a functional termination sequence in the same host organism.

The invention also relates to a vector comprising a polynucleotideaccording to the invention and/or an expression cassette according tothe invention.

The invention also relates to a host organism transformed with apolynucleotide according to the invention, an expression cassetteaccording to the invention and/or a vector according to the invention.

The invention also relates to compositions for use as a drug comprisinga polypeptide according to the invention, a polynucleotide according tothe invention, an expression cassette according to the invention, avector according to the invention and/or a host organism according tothe invention.

In a preferred embodiment, the invention relates to compositions for thetreatment of thrombotic diseases.

In another preferred embodiment, the invention relates to compositionsfor the treatment of disorders of haemostasis.

The invention also relates to the use of these compositions as acicatrisation agent.

Lastly, the invention relates to cosmetic compositions comprising apolypeptide such as described above.

The invention relates to isolated polypeptides having the sequence ofthe polypeptide of SEQ ID No. 1 or the polypeptide of position 25 toposition 152 of SEQ ID No. 1.

The peptide of position 1 to position 24 of SEQ ID No. 1 corresponds tothe signal peptide that enables secretion of the recombinant protein bya host cell. This signal peptide can be absent or be replaced by anothersignal peptide according to techniques well known to the person skilledin the art. The person skilled in the art will be able to choose thehomologous or heterologous signal peptide suitable for the expressionand secretion of the polypeptides of the present invention in variousprokaryotic or eukaryotic expression systems. Preferably, thepolypeptides of the present invention are produced in eukaryotic cellsor organisms and in particular in mammalian cells. In a particularembodiment of the invention, the polynucleotides of the presentinvention comprise a signal peptide enabling their secretion in theextracellular medium. In another embodiment, the invention relates tothe mature polypeptide obtained after cleavage of the signal peptide.

The polypeptides of the present invention have a biological activity andnotably a pro-aggregating activity on human blood platelets detectedusing a platelet aggregometer (Regulest, Florange, France), according tothe reference technique (Born, 1962). Platelet-rich plasma (PRP) isbrought into contact with an agonist (10 μl in 290 μl PRP) and theclearing of the medium (related to the formation of aggregates whichfall to the bottom of the tube) is monitored in real time (aggregationcurve). In the absence of platelet aggregation, the signal remains flat(persistent turbid medium). It is possible to verify the absence orpresence of aggregates by examination of the tube at the end ofmeasurement. Evaluation of the platelet aggregation test response iscarried out at 37° C., with continuous stirring (1000 rpm). Theapparatus is calibrated as follows: 0% aggregation with platelet-richplasma (preparation obtained by slow centrifugation and concentrationadjusted to 300×10⁹/L) and 100% aggregation with platelet-poor plasma(preparation obtained by rapid centrifugation). The quality of theplatelet preparations was validated by verifying the response toreference agonists (5 μM ADP and 1 μg/ml collagen), used for thedevelopment of antiplatelet therapeutics. A protein is consideredpro-aggregating when it is capable of inducing aggregation greater than30%, and in an irreversible way.

The receptor recognition peptide motifs expressed on the surface of manycell types confer on the polypeptide activity of the following types:

-   -   cell adhesion,    -   cell recruitment.

The pro-aggregating effect of the polypeptide, independent of its triplehelix structure, is the consequence of the activation of one or morereceptors present on the platelet surface (α1β2, TIIICBP and GPVI).

The invention also relates to fragments of the polypeptide of SEQ ID No.1 that preserve at least one of the activities of the polypeptide of SEQID No. 1. The term “fragment” of a polypeptide indicates a polypeptidecomprising a part but not the totality of the polypeptide from which itis derived. The invention thus relates to a polypeptide comprising afragment of at least 100, 110, 120, 130, 140 or 150 amino acids of thepolypeptide of SEQ ID No. 1.

These fragments of the polypeptide of SEQ ID No. 1 preserve at least oneof the activities of the polypeptide of SEQ ID No. 1, in particularpro-aggregating activity on human blood platelets. The invention thusrelates to the biologically active fragments of the polypeptide of SEQID No. 1. The term “biologically active fragment” indicates a fragmentof a polypeptide that preserves the function of the polypeptide fromwhich it is derived. The biologically active fragments of thepolypeptide of SEQ ID No. 1 thus preserve at least one of the functionsof this polypeptide and preferably preserve all the biologicalactivities of the polypeptide of SEQ ID NO. 1. The methods for preparingfragments of a polypeptide as well as the techniques for measuring thebiological activities of the polypeptides of the present invention arewell known to the person skilled in the art.

The invention also relates to polypeptides that have at least one of theactivities of the polypeptide of SEQ ID No. 1 and with amino acids atleast 70% identical to the polypeptide of SEQ ID No. 1. Preferably,these polypeptides have the same properties and notably the samebiological activities as the polypeptide of SEQ ID No. 1. The inventionrelates to polypeptides with amino acids at least 70%, 80%, 90%, 95%,98% and preferentially at least 99% identical to the polypeptide of SEQID No. 1. “Identical amino acids” means amino acids that are invariantor unchanged between two sequences. These polypeptides can have adeletion, an addition or a substitution of at least one amino acidcompared to the polypeptide of SEQ ID No. 1.

The invention also relates to polypeptides with at least 70%, 80%, 90%,95%, 98% and preferentially at least 99% homology with the polypeptideof SEQ ID No. 1. “Homology” means the measurement of the resemblancebetween protein sequences. These polypeptides can have a deletion, anaddition or a substitution of at least one amino acid compared to thepolypeptide of SEQ ID No. 1. The degree of homology between twosequences, quantified by a score, is based on the percentage ofidentities and/or preserving substitutions of the sequences.

The polypeptides of the present invention with a certain degree ofhomology or identity with the polypeptide of SEQ ID No. 1 comprise atleast 100 or 150 amino acids.

Methods for measuring and identifying the degree of identity and thedegree of homology between polypeptides are known to the person skilledin the art. The AlignX alignment tool (Clustal W algorithm) of theVector NTi 9.1.0 software package (Invitrogen INFORMAX,http://www.invitrogen.com), for example, can be used, preferably usingthe default settings.

The polypeptides according to the invention are isolated or purifiedfrom their natural environment. The polypeptides can be prepared bymeans of various methods. These methods are notably production ofrecombinant polypeptides by suitable host cells and their subsequentpurification, production by chemical synthesis or, finally, acombination of these various approaches. These various productionmethods are well known to the person skilled in the art. Preferably, thepolypeptides of the present invention are produced by recombinantprokaryotic or eukaryotic cells. The polypeptides of the presentinvention can thus be produced in bacteria or in mammalian cells.

The invention also relates to fusion proteins, proteins or chimericproteins comprising the polypeptides according to the invention. Theterm “polypeptide” indicates proteins as well as modified polypeptides.

In one embodiment of the invention, the polypeptides according to theinvention are glycosylated. The polypeptide of SEQ ID No. 1 has notablyO-glycosylation sites on the lysine amino acids present in positions 102and 141 (in position 3 of a GXY triplet). In a preferred embodiment, theasparagine residue in position 93 of the polypeptide of SEQ ID No. 1 isglycosylated.

The invention also relates to polynucleotides coding for thepolypeptides defined above. According to the present invention,“polynucleotide” means a single-strand nucleotide chain or its DNA orRNA complement, or a double-strand nucleotide chain which can be ofcomplementary or genomic DNA. Preferably, the polynucleotides of theinvention are DNA, in particular double-strand DNA. The term“polynucleotide” also means modified polynucleotides. Thepolynucleotides of the present invention are isolated or purified fromtheir natural environment. Preferably, the polynucleotides of thepresent invention can be prepared by the classic molecular biologytechniques as described by Sambrook et al. (Molecular Cloning: ALaboratory Manual, 1989) or by chemical synthesis.

In a first embodiment, the invention relates to the polynucleotide ofSEQ ID No. 2. In a second embodiment, the invention relates to thepolynucleotide of SEQ ID No. 2 whose sequence lies between position 73and position 459 of SEQ ID No. 1. These polynucleotides code for thepolypeptides defined above.

The invention also relates to polynucleotides with at least 70%, 75%,80%, 85%, 90%, 95%, 98% and preferably at least 99% identity with thepolynucleotide of SEQ ID No. 2 or with the polynucleotide whose sequencelies between position 73 and position 459 of SEQ ID No. 2. Thesepolynucleotides code preferably for a polypeptide that preserves thebiological activities of the polypeptide of SEQ ID No. 1.

“Identical nucleotides” means nucleotides that are invariant orunchanged between two sequences. These polynucleotides can have adeletion, an addition or a substitution of at least one nucleotidecompared to the reference polynucleotide.

The invention also relates to polynucleotides with at least 70%, 75%,80%, 85%, 90%, 95%, 98% and preferably at least 99% homology with thepolynucleotide of SEQ ID NO. 2 or with the polynucleotide whose sequencelies between position 73 and position 459 of SEQ ID No. 2. Thesepolynucleotides code preferably for a polypeptide that preserves thebiological activities of the polypeptide of SEQ ID No. 1.

“Homology” means the measurement of the resemblance between nucleicsequences. These polynucleotides can have a deletion, an addition or asubstitution of at least one nucleotide compared to the referencepolynucleotide. The degree of homology between two sequences, quantifiedby a score, is based on the percentage of identities and/or preservingsubstitutions of the sequences.

Methods for measuring and identifying the degree of identity and thedegree of homology between nucleic acid sequences are well known to theperson skilled in the art. The AlignX alignment tool (Clustal Walgorithm) of the Vector NTi 9.1.0 software package (InvitrogenINFORMAX, http://www.invitrogen.com), for example, can be used,preferably using the default settings.

The invention also relates to polynucleotides capable of hybridisingselectively with the polynucleotide of SEQ ID NO. 2 or with thepolynucleotide whose sequence lies between position 73 and position 459of SEQ ID No. 2. Preferably, selective hybridisation is carried outunder conditions of medium stringency and preferentially underconditions of high stringency. These polynucleotides code preferably forpolypeptides having the biological activities of the polypeptide of SEQID No. 1. “Sequence capable of hybridising selectively” means, accordingto the invention, the sequences that hybridise with the referencesequence at a level significantly greater than the background noise. Thesignal level generated by the interaction between the sequence capableof hybridising selectively and the reference sequences is generally 10times more intense, preferably 100 times more intense than that of theinteraction of the other DNA sequences generating the background noise.The stringent hybridisation conditions that enable selectivehybridisation are well known to the person skilled in the art. Ingeneral, the hybridisation and washing temperature is at least 5° C.lower than the Tm of the reference sequence at a given pH and for agiven ionic strength. Typically, the hybridisation temperature is atleast 30° C. for a polynucleotide of 15 to 50 nucleotides and at least60° C. for a polynucleotide of more than 50 nucleotides. For example,hybridisation is carried out in the following buffer: 6×SSC, 50 mMTris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, 500μg/ml denatured salmon sperm DNA. Washings, for example, are carried outsuccessively with low stringency in 2×SSC, 0.1% SDS buffer, with mediumstringency in 0.5×SSC, 0.1% SDS buffer and with high stringency in0.1×SSC, 0.1% SDS buffer. Hybridisation can of course be carried outaccording to other common methods well known to those persons skilled inthe art (see in particular Sambrook et al., Molecular Cloning: AtLaboratory Manual, 1989). Preferably, the polynucleotides that hybridiseselectively to a reference polynucleotide preserve the function of thereference sequence.

The invention generally relates to polynucleotides coding for thepolypeptides according to the invention. Due to degeneration of thegenetic code, different polynucleotides can code for the samepolypeptide.

The invention also relates to an antibody binding specifically to thepolypeptide of SEQ ID No. 1.

“Binding specifically” means that these antibodies bind only to thepolypeptide of SEQ ID No. 1. In particular, the antibodies do not bindto other antigens and notably not to other collagenous proteins.

The antibodies according to the present invention are preferablyspecific monoclonal antibodies, notably of murine origin, chimeric orhumanised which could be obtained according to standard methods wellknown to the person skilled in the art.

In general, the techniques for preparing monoclonal antibodies or theirfunctional fragments, notably of murine origin, can be selected fromthose described in particular in the handbook Antibodies (Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor N.Y., p. 726, 1988) or the hybridoma-basedpreparation technique well known to the person skilled in the art.“Antibody” also means chimeric or humanised antibodies.

The invention also relates to expression cassettes comprising in thedirection of transcription:

-   -   a functional promoter in a host organism,    -   a polynucleotide according to the invention,    -   a functional termination sequence in the same host organism.

According to one embodiment of the invention, a polynucleotide codingfor a polypeptide according to the invention is inserted in anexpression cassette by using cloning techniques well known to the personskilled in the art. This expression cassette comprises the elementsnecessary to the transcription and translation of sequences coding forthe polypeptides according to the invention.

Advantageously, this expression cassette comprises at the same timeelements that cause a host cell to produce a polypeptide and elementsnecessary to the regulation of this expression.

Any type of promoter sequence can be used in the expression cassettesaccording to the invention. The choice of promoter will depend notablyon the host organism chosen for the expression of the gene of interest.Certain promoters enable constitutive expression whereas other promotersare on the contrary inducible. Among the functional promoters inbacteria, that of the bacteriophage T7 RNA polymerase can be cited inparticular. Among the functional promoters in yeasts, the GAL1 genepromoter or the GAL4 and ADH promoters of S. cerevisiae can be cited.Among the functional promoters in higher eukaryotic cells and notably inmammalian cells, CMV (cytomegalovirus), SV40, RSV (Rous sarcoma virus),human or chicken beta-actin, beta-globin, PGK (phosphoglycerate kinase),EF1alpha, thymidine kinase, and MMTV (mouse mammary tumour virus) can becited. All these promoters are described in the literature and are wellknown to the person skilled in the art.

Expression cassettes, according to the present invention, can furtherinclude any other sequence necessary to the expression of polypeptidesor polynucleotides such as, for example, regulation elements or signalsequences enabling the secretion of polypeptides produced by the hostorganism. Any regulation sequence that increases the level of expressionof the coding sequence inserted in the expression cassette can benotably used. According to the invention, other regulation sequences,which are located between the promoter and the coding sequence, such astranscription activators (“enhancers”), can notably be used incombination with the promoter regulation sequence.

A wide variety of termination sequences can be used in the expressioncassettes according to the invention; these sequences enable thetermination of transcription and mRNA polyadenylation. Any functionaltermination sequence in the selected host organism can be used.

The present invention also relates to a polynucleotide comprising anexpression cassette according to the invention, advantageously theexpression cassettes according to the present invention are inserted ina vector.

The present invention thus also relates to replication or expressionvectors for the transformation of a host organism comprising at leastone polynucleotide or expression cassette according to the presentinvention. This vector can notably correspond to a plasmid, a cosmid, abacteriophage or a virus in which is inserted a polynucleotide or anexpression cassette according to the invention. The techniques forconstructing these vectors and for inserting in these vectors apolynucleotide of the invention are well known to the person skilled inthe art. Generally, any vector capable of being maintained, capable ofself replicating or capable of propagating in a host cell in order toinduce notably the expression of a polynucleotide or a polypeptide canbe used. The person skilled in the art will choose suitable vectorsaccording to the host organism to be transformed, and according to thetransformation technique used.

The vectors of the present invention are notably used to transform ahost organism with a view to the replication of the vector and/or theexpression of a polypeptide according to the invention in the hostorganism.

The invention also relates to a method for preparing a polypeptideaccording to the invention comprising the following steps:

-   -   a host organism is transformed with an expression vector        comprising an expression cassette according to the invention        and/or with a polynucleotide according to the invention,    -   the polypeptides produced by the host organism are isolated.

The present invention also relates to a method for transforming a hostorganism by integrating in the aforementioned host organism at least onepolynucleotide or expression cassette or vector according to theinvention. The polynucleotide can be integrated in the genome of thehost organism or replicated in a stable manner in the host organism.Methods for transforming host organisms are well known to the personskilled in the art and are widely described in the literature.

The present invention also relates to a host organism transformed with apolynucleotide, expression cassette or vector according to theinvention. “Host organism” means in particular according to theinvention any unicellular or multicellular, lower or higher organism, inparticular selected from bacteria, yeasts and cells of highereukaryotes, in particular mammalian cells such as CHO (Chinese hamsterovary cells), BHK (baby hamster kidney), HEK-293 (human embryonic kidneycell line), NSO (mouse myeloma cell line), Per.C6 (Crucell), and YB2/0(ATCC no. CRL 1662). “Host organism” means a nonhuman organism.

The invention also relates to compositions for use as a drug comprisinga polypeptide according to the invention, a polynucleotide according tothe invention, an expression cassette according to the invention, avector according to the invention and/or a host organism according tothe invention. The invention thus also relates to pharmaceuticalcompositions comprising a polypeptide according to the invention, apolynucleotide according to the invention, an expression cassetteaccording to the invention, a vector according to the invention and/or ahost organism according to the invention.

In a preferred embodiment, the invention relates to compositions for theprevention and/or the treatment of thromboses.

In another embodiment, the invention relates to compositions for theprevention or the treatment of disorders of haemostasis.

The invention also relates to said compositions for use as acicatrisation agent.

In another embodiment, the inventive compositions can also be used as:

-   -   diagnostic reagents for detecting:        -   certain platelet dysfunctions        -   haematological diseases    -   active components of haemostatic bandages and adhesives in the        composition of dermatological and cosmetic creams.

The invention also relates to therapeutic methods for treatingthromboses comprising the administration to an individual of aneffective quantity of a polypeptide according to the invention, apolynucleotide according to the invention, an expression cassetteaccording to the invention, a vector according to the invention and/or ahost organism according to the invention.

The invention relates lastly to the use of the polypeptides,polynucleotides and transformed host organisms of the present inventionfor the manufacture of drugs.

The invention relates to pharmaceutical compositions comprising apolypeptide, polynucleotide or transformed host organism such as definedin the present invention and a suitable pharmaceutical excipient.

Said compositions can be formulated for administration to mammals,including humans. The dosing regimen varies according to the treatmentand the disease in question. Said compositions are prepared in such away as to be capable of being administered by the digestive orparenteral route.

In the pharmaceutical compositions of the present invention for oral,sublingual, subcutaneous, intramuscular, intravenous, transdermal, localor rectal administration, the active ingredient can be administered inunit forms of administration, in mixture with classic pharmaceuticalcarriers, to animals or humans. Suitable unit forms of administrationcomprise forms by oral route such as tablets, gelatin capsules, powders,granules and oral solutions or suspensions; sublingual and buccal formsof administration; subcutaneous, intramuscular, intravenous, intranasalor intraocular forms of administration; and rectal forms ofadministration.

When a solid composition in tablet form is prepared, the principalactive ingredient is mixed with a pharmaceutical excipient such asgelatin, starch, lactose, magnesium stearate, talc, gum arabic oranalogues. Tablets can be coated with saccharose or other suitablematerials or can be treated in such a way that they have extended ordelayed activity and that they continuously release a predeterminedquantity of active ingredient.

A preparation in gelatin capsules is obtained by mixing the activeingredient with a diluent and by pouring the mixture obtained into softor hard gelatin capsules.

A preparation in syrup or elixir form can contain the active ingredienttogether with a sweetener, an antiseptic, as well as a flavouring agentand a suitable colorant.

Water-dispersible powders or granules can contain the active ingredientin mixture with dispersion or wetting agents, or suspension agents, aswell as with flavour correctors or sweeteners.

The invention also relates to cosmetic compositions comprising apolypeptide as described above.

These compositions can moreover comprise active ingredients classicallyused in dermatology such as emollients, hydrating active ingredients,cutaneous barrier restructuring agents, anti-irritants, and soothingagents. The cosmetic compositions according to the invention can beformulated in the form of various preparations suitable for topicalapplication. According to a preferred embodiment, the variouspreparations are suitable for topical application and include creams,emulsions, milks, pomades, lotions, oils, aqueous or hydro-alcohol orglycol solutions, powders, sprays, jellies, gels, hydrogels or any otherproduct for external application. These cosmetic compositions can alsocontain antioxidants, preservatives, etc. The invention further relatesto a method of cosmetic treatment, hygienic care, or embellishmentand/or a method to perfume mucosa and/or skin that is normal, dry, oily,mixed, dehydrated, aged, sensitive, irritated, uncomfortable,intolerant, with an imbalance related to intrinsic, extrinsic orhormonal ageing or related to exogenic attack (pollutants, UV, stress,etc.), with allergic tendency, or with pigmentation disorders,characterised in that it consists in applying a composition according tothe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Expression of messenger RNA coding for the protein in adherentCHO-S cells transfected in a transitory fashion.

FIG. 2: FIG. 2 represents platelet aggregation induced by theconditioned medium (CM) of adherent CHO-S cells, transfected in atransitory fashion, in the presence or absence of epinephrine.

FIG. 3: FIG. 3 represents expression of messenger RNA coding for theprotein in a stable cell clone.

FIG. 4: FIG. 4 shows detection by Western blotting of the presence ofprotein-6X-histidine in the various bacterial fractions obtained afterpurification.

FIG. 5: FIG. 5 shows platelet aggregation induced by the proteinproduced in bacteria.

FIG. 6: FIG. 6 shows validation by Western blotting of the specificityof the rabbit serum obtained after 89 days of immunisation.

EXAMPLES

A. Transitory Production by Mammalian Cells

1. Vector Construction

Plasmid 1 (NVH001-A)

Plasmid NVH001-A contains the following elements:

-   -   an intron from plasmid pCIneo (Promega: pCIneo Mammalian        Expression Vector)    -   the inventive protein NVH001 from vector pUC57-NVH001 (ordered        from GeneCust) and possessing type-III collagen signal peptide        (NP_(—)000081)    -   a polyA tail from hGH mRNA (NM_(—)000515)    -   a CMV promoter from pCDF1-MCS1-EF1-copGFP (System Biosciences:        pCDF cDNA Cloning and Expression Lentivectors).

Plasmid 2 (NVH001-B1), 3 (NVH001-B2), 4 (NVH001-C1) and 5 (NVH001-C2)

Plasmids NVH001-B1 and NVH001-C1 contain the following elements:

-   -   an SV40 origin from derived plasmid pSV2-Neo (ori SV40:1-989 in        vector ATCC 37149)    -   a promoter/enhancer that can be one of those described in        Molecular Cloning: A Laboratory Manual: 3rd edition, Sambrook        and Russell, 2001, Cold Spring Harbor Laboratory Press    -   a hygromycin cassette from pMono-hygro-mcs (Invitrogen)    -   the inventive protein NVH001 from vector pUC57-NVH001 (ordered        from GeneCust) and possessing type-III collagen signal peptide        (NP_(—)000081)    -   a C-propeptide from plasmid pUC57-proC (ordered from GeneCust)    -   a polyA tail from hGH mRNA (NM_(—)000515).

Plasmids NVH001-B2 and NVH001-C2 contain the same elements as plasmidsNVH001-B1 and C1 except C-propeptide.

All the final plasmids are sequenced to verify that no mutation wasintroduced in the DNA of the protein of interest and in the elementsprovided.

2. Transfection of Mammalian Cells

Plasmid 1 (NVH001-A)

1. Vector NVH001-A is transfected, in a transitory fashion, in CHO-Scells made adherent and cultivated in RPMI medium containing 5% foetalcalf serum using the Lipofectamine 2000 kit from Invitrogen.

2. The culture supernatants are collected at days 3 and 4, the proteinsproduced are purified and then their platelet binding activity is testedusing an aggregometer.

Plasmids 2 (NVH001-B1), 3 (NVH001-B2), 4 (NVH001-C1) and 5 (NVH001-C2)

1. Vectors NVH001-B1, NVH001-B2, NVH001-C1 and NVH001-C2 are transfectedin a transitory fashion in CHO-S cells made adherent and cultivated inRPMI medium containing 5% foetal calf serum using the Lipofectamine 2000kit from Invitrogen.

The RNA of the transfected cells are extracted at days 1, 2, 3, 4 and 5by the adding of 800 μl of TRIZOL (Invitrogen). The corresponding cDNAare obtained by reverse transcription from 1 μg total RNA. Amplificationby PCR was carried out on the cDNA. Amplicons are then detected aftermigration on a 2% agarose gel for 30 minutes at 125 V. A positivecontrol (transfected plasmid) and a negative control were also carriedout. The molecular weight marker (at left) is used to verify ampliconsize.

Expression in the cells transfected with mRNA coding for the inventiveprotein is observed as of day 1. This expression increases slightly overtime and is maintained after 5 days. The progress of mRNA expression isrepresented in FIG. 1.

2. The culture supernatants are collected at days 3 and 4, the proteinsproduced are purified and then their platelet binding activity is testedusing an aggregometer.

3. Biological Activity of the Inventive Protein: Platelet Aggregation

The platelet binding activity of the inventive protein is evaluated byaggregometry. Blood samples in citrate tubes, collected from healthyvolunteer donors after signed consent, are centrifuged at 150 g for 15minutes in order to obtain platelet-rich plasma (PRP). Concentration isadjusted to 300 giga/L. Aggregation tests are carried out using aRegulest® platelet aggregometer, with stirring (1000 rpm) at 37° C. Thistechnique is based on photometric reading according to the Born methodwhich consists in measuring the evolution of the light transmission of aturbid medium, PRP, in response to a platelet function activator. Ifplatelet aggregates are formed, the medium clears up and an increase inlight transmission is observed. A molecule is considered to have apro-aggregating effect when an increase of more than 30% is observed inthis transmission associated with a break in the slope corresponding tothis clearing.

FIG. 2 represents platelet aggregation induced by the conditioned mediumof adherent CHO-S cells, transfected in a transitory fashion, in thepresence or absence of epinephrine.

The reaction mixture is composed of 290 μl PRP to which is added 30 μlconditioned medium in the presence or absence of 0.1 μM epinephrine.Platelet response is measured for 10 minutes. Self-aggregation (-∘-)defines the baseline. Adding 0.1 μM epinephrine induces a slight shiftof the baseline, stable for 10 minutes (-●-). Adding 30 μl conditionedmedium induced a limited response (18%) after 2 minutes (-∇-). Thisshift is stable during the 10 minutes of observation. Adding epinephrinefor 2 minutes, followed by adding 30 μl conditioned medium induced totalplatelet aggregation in 5 minutes (-▾-). This aggregation is notreversible, suggesting that the aggregates formed are stable.

Adding 30 μl of conditioned medium of non-transfected adherent CHO-Scells does not induce aggregation (result not presented).

B. Stable Production by Mammalian Cells

1. Vector Construction

Plasmid 1 (NVH001-A)

1. The intron-NVH001-hGHpA unit is removed from the vector constructedfor transitory expression and is cloned in a production vectorcontaining a hygromycin cassette from pMono-hygro-mcs (Invitrogen).

2. The final vector is sequenced to verify that no mutation wasintroduced in the DNA of the protein of interest and in the elementsprovided.

3. The linearised vector is transfected in a stable fashion in CHO cellsmade adherent using the Lipofectamine 2000 kit from Invitrogen. Thetransfected cells are selected by progressive levels of hygromycin B.

FIG. 3 represents expression of messenger RNA coding for the protein ina stable cell clone.

Cell RNA are extracted at days 1, 2, 3 and 4 by the adding of 800 μlTRIZOL (Invitrogen). The corresponding cDNA are obtained by reversetranscription from 1 μg total RNA. PCR amplification was carried out oncDNA using 5′-AGCTGGCGCGCCGCCACCATG-3′ (S) and5′-GCTTCCGGGAGGCCCTGGCTTCCCATC-3′ (AS) primers. Amplicons are thendetected after migration on a 2% agarose gel for 30 minutes at 125 V. Apositive control, corresponding to plasmid DNA, is used. The molecularweight marker (at right) is used to verify amplicon size. Stableexpression is observed in the cell clone.

C. Production in Bacteria

1. Construction

The DNA of the molecule of interest (NVH001) is amplified by PCR on thevector pUC57-NVH001 using the tagNVH001′-sense primer:GCTGCCATGGGCAGCAGCCATCATCATCATCATCACGGTCGCCCGGGAGCTCCTGGAGAGA GAGGATTGand the tagNVH001′-antisense primer:GCACGGATCCTATTAGCCAGGGCAAGGTCCAGGGGCTC which makes it possible to add a6X-histidine tag on the N-terminal side of protein NVH001. The PCRproducts are cloned in vector pET3d (pET3d-NVH001) enabling productionof the molecule in bacteria (New England Biolab).

The presence and the correct sequence of the DNA are verified bysequencing.

2. Production

1. The vector pET3d-NVH001 is transfected in BL21 bacteria enabling themass production of protein.

2. The bacteria are inoculated in 1 l nutritive medium and are incubatedat 37° C. until OD_(600nm) is obtained in which the bacteria are inexponential phase.

3. Protein production is induced by IPTG for 5 hours at 30° C.

4. The bacteria are pelleted, lysed and the inventive protein ispurified in the soluble (supernatant) and insoluble (inclusion body)fraction of the bacteria on Macherey-Nagel anti-6X-histidine tagcolumns.

FIG. 4 shows detection by Western blotting of the presence ofprotein-6X-histidine in the various bacterial fractions obtained afterpurification.

40 μl of each fraction obtained after elution of the anti-6X-histidinetag column is deposited on a 16% polyacrylamide gel. Electrophoresis iscarried out in migration buffer (192 mM glycine, 25 mM Tris-HCl pH 6.8and 0.1% SDS) at 18 mA, in the presence of molecular weight markers(Amersham) using the Mini-Protean 3 migration system (Biorad). Theproteins separated in the gel are transferred on a PVDF membrane(Biorad) for 60 min at 70 V. Non-specific binding is blocked byincubation of the membrane for 120 min at 37° C. and with stirring, in asaline solution of Tris+0.1% Tween-20 (TBS-T) containing 5% skimmedmilk. The membrane is then incubated overnight at 4° C. with ananti-6X-histidine tag monoclonal antibody (Cell Signaling) diluted to1/1000 in TBS-T. After 3 washings of 5 min in TBS-T, the membrane isincubated for 60 min at room temperature in an anti-mouse antibodycoupled with peroxidase (Jackson Laboratories) diluted to 1/10,000. Themembrane is again washed three times for 5 min in TBS. The result isvisualised by the chemiluminescence technique using the ECL kit(Amersham).

The presence of protein is observed in the various fractions. Theprincipal band obtained with a molecular weight of roughly 19 kDacorresponds to the monomeric form of the protein. Bands can also beobserved in certain fractions corresponding to proteins with molecularweights of 26 kDa, 43 kDa and 60 kDa.

3. Demonstration of its Biological Activity: Platelet Aggregation

The platelet binding activity of the inventive protein is evaluated byaggregometry. Blood samples in citrate tubes, collected from healthyvolunteer donors after signed consent, are centrifuged at 150 g for 15minutes in order to obtain platelet-rich plasma (PRP). The concentrationis adjusted to 300 giga/L. Aggregation tests are carried out using aRegulest® platelet aggregometer, with stirring (1000 rpm) at 37° C. Thistechnique is based on photometric reading according to the Born methodwhich consists in measuring the evolution of the light transmissionthrough the PRP, which begins as a turbid medium, in response to aplatelet activator. If platelet aggregates are formed, the medium clearsup and an increase in light transmission is observed. A molecule isconsidered to have a pro-aggregating effect when an increase of morethan 30% is observed in this transmission associated with a break in theslope corresponding to this clearing.

FIG. 5 shows platelet aggregation induced by the protein produced inbacteria.

The various fractions eluted from the anti-6X-histidine tag columncontaining the protein detected by Western blotting are collected and alyophilisate is obtained by centrifugation. Part of this lyophilisate isincubated in the presence of 0.25% glutaraldehyde for 3 hours at 4° C.and then dialysed overnight against 1×PBS at 4° C. before its use inaggregometry.

The reaction mixture is composed of 290 μl PRP to which is added 30 μlPBS containing the protein. Platelet response is measured for 25minutes. The lines marked -∘- and -∇- show the response obtained afterthe adding of a solution of PBS and of dialysed PBS-glutaraldehyde,respectively: no aggregation is observed. The adding of PBS containingthe protein not aggregated by glutaraldehyde (-▾-) or aggregated byglutaraldehyde and dialysed (-●-) shows significant aggregation 10minutes after they were added. This aggregation reaches 90% (-▾-) and100% (-●-), respectively, after 20 minutes.

D. Production of Anti-Protein of Interest Antibody in Rabbit

1. The protein is produced in large quantity in BL21 bacteria aspreviously described.

2. Two rabbits are immunised 4 times at 3-week intervals, that is to sayat D0, D21, D42 and D63, with the purified protein supplemented (v/v)with Freund's adjuvant.

3. The rabbits are sacrificed at the end of the protocol at D89 and thesera are recovered by centrifugation after exsanguination of theanimals.

4. The IgG are purified by affinity chromatography on a specific columnin order to eliminate plasma proteins.

FIG. 6 shows the validation by Western blotting of the specificity ofthe rabbit serum obtained after 89 days of immunisation.

10 μl of two preparations of proteins produced in bacteria, whoseaggregating activity was validated by aggregometry, is deposited on a16% polyacrylamide gel. Electrophoresis is carried out in migrationbuffer (192 mM glycine, 25 mM Tris-HCl pH 6.8 and 0.1% SDS) at 18 mA, inthe presence of molecular weight markers (Amersham) using theMini-Protean 3 migration system (Biorad). The proteins separated in thegel are transferred on a PVDF membrane (Biorad) for 60 min at 70 V.Non-specific binding is blocked by incubation of the membrane for 120min at 37° C. and with stirring, in a saline solution of Tris+0.1%Tween-20 (TBS-T) containing 5% skimmed milk. The membrane is thenincubated overnight at 4° C. either with rabbit serum, withdrawn at D89of the immunisation protocol and diluted to 1/100, or with ananti-6X-histidine tag monoclonal antibody (Cell Signaling) diluted to1/1000 in TBS-T. After 3 washings of 5 min in TBS-T, the membrane isincubated for 60 min at room temperature in an anti-mouse or anti-rabbitantibody coupled with peroxidase (Jackson Laboratories) diluted to1/10,000. The membrane is again washed three times for 5 min in TBS. Theresult is visualised by the chemiluminescence technique using the ECLkit (Amersham).

Visualisation shows the presence of a double band, present in the twoprotein preparations (A, wells 1 and 2), with a molecular weight of 60kDa. This band is also detected by the anti-6X-histidine tag antibody,showing that it is indeed the inventive protein (B, wells 1 and 2). Itshould be noted that we no longer observe the other bands detected bythe anti-6X-histidine tag antibody on the purified fractions ofbacteria. This result suggests that immunisation was only effective forthe multimeric form of the inventive protein or that the inventiveprotein assembles into a multimer once purified.

References

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The invention claimed is:
 1. An isolated polypeptide wherein saidpolypeptide has the sequence of the polypeptide of SEQ ID NO:1 or thepolypeptide of position 25 to position 152 of SEQ ID NO:1.
 2. A drugcomposition comprising a polypeptide according to claim
 1. 3. Thecomposition according to claim 2 for the treatment of thromboticdiseases.
 4. The composition according to claim 2 for the treatment ofdisorders of haemostasis.
 5. The composition according to claim 2 foruse as a cicatrisation agent.
 6. A cosmetic composition comprising thepolypeptide according to claim 1.